Biomolecules Responsible for the Total Antioxidant Capacity (TAC) of Human Plasma in Healthy and Cardiopathic Individuals: A Chemical Speciation Model
Round 1
Reviewer 1 Report
line 111 - preparation of solution must be describe
line 190 - please desribe redox reaction
references 5, 6, 9 - must have DOI
Author Response
Comments and Suggestions for Authors
line 111 - preparation of solution must be describe
Response: now lines 112-113 - Details for the solutions preparation used in the TAC determination are fully described in ref. 11 and are omitted in this work in order to avoid redundant information.
line 190 - please describe redox reaction
Response: lines 190-197: description was provided
references 5, 6, 9 - must have DOI
Response: now references 6, 7, 10: the DOI were added, except that of reference 9 because it is antecedent to 2000.
Reviewer 2 Report
The research article, entitled "Biomolecules responsible of the Total Antioxidant Capacity (TAC) of human plasma in healthy and cardiopathic individuals: a chemical speciation model” aims to improve the information on the extracellular redox buffer capacity of the organism. In addition, this model allows i) identifying the biomolecules responsible of the redox buffer capacity in the human plasma, ii) identifying the weight of each redox active biomolecule in determining different homeostasis, and iii) fractioning the TAC between food and metabolic sources, iv) opening a way of investigation regarding the response to oxidative stress conditions in terms of extracellular redox reactivity based on a modelling approach. The scientific manuscript is very interesting, however, some problems, as indicated below, should be addressed before the document can be considered for publication in this journal. This version of the manuscript is not enough complete.
Minor problems:
English language and style are fine, minor spell check is required to ensure that an international audience can clearly understand your text.
Major problems:
I suggests to improve the introduction section. Oxidative stress is frequently described as an imbalance between production of reactive oxygen species in biological systems, and their ability to defend through the sophisticated antioxidant machinery. Is there an interplay between Total Antioxidant Capacity and increase of oxidative stress levels? The authors should add other information about this phenomenon, especially improve the clinical meaning and utility of TAC.
Why the choice of this category of patients? Could it depend by disease?
Plasma is the target of this investigation. It is mostly water, and contains important proteins, glucose, clotting factors, electrolytes, hormones, vitamins, oxidants, carbon dioxide and oxygen. In this context, it plays a vital role for measuring the TAC efficiency. However, some of these molecules (vitamins or oxidants), transported by the blood stream, could interact with erythrocytes plasma membrane structures, thus causing potential consequences on erythrocytes (the most numerous cells in the blood) . How do the authors explain this? Could the measure be changed? The organism aims to maintain a beneficial chemical equilibrium to perform specific biological functions, being a method depending quantity, the concentration of the plasma redox active biomolecules could be altered? I suggest to add this aspect in your study, and to examine the following papers (10.3390/antiox9010025 et al.,), in which the authors underscore the role of different substance in erythrocytes homeostasis, as well as the potential antioxidant effects. It is possible that there is not a direct ratio between TAC efficiency and specific disease. In fact, the TAC is major in cardiopathic individuals compared to the control, and these findings could explain the capacity of the organism to defend by reactive species. The authors should also analyze this aspect.
Plasma from 25 cardiovascular surgery patients –17 males and 8 females– was collected and subjected to the measurements, data treatments and modelling as those of the healthy individuals (85 blood donors – 47 males and 38 females). Is this the correct number? The number of cardiovascular surgery patients could be very low compared to healthy volunteers in order to analysis. The authors should explain the choice.
Author Response
Comments and Suggestions for Authors
The research article, entitled "Biomolecules responsible of the Total Antioxidant Capacity (TAC) of human plasma in healthy and cardiopathic individuals: a chemical speciation model” aims to improve the information on the extracellular redox buffer capacity of the organism. In addition, this model allows i) identifying the biomolecules responsible of the redox buffer capacity in the human plasma, ii) identifying the weight of each redox active biomolecule in determining different homeostasis, and iii) fractioning the TAC between food and metabolic sources, iv) opening a way of investigation regarding the response to oxidative stress conditions in terms of extracellular redox reactivity based on a modelling approach. The scientific manuscript is very interesting, however, some problems, as indicated below, should be addressed before the document can be considered for publication in this journal. This version of the manuscript is not enough complete.
Answer: We thank the reviewer for the kind comments. The answers to each comment are reported below.
Minor problems:
English language and style are fine, minor spell check is required to ensure that an international audience can clearly understand your text.
Answer: The spell check was done.
Major problems:
I suggests to improve the introduction section. Oxidative stress is frequently described as an imbalance between production of reactive oxygen species in biological systems, and their ability to defend through the sophisticated antioxidant machinery. Is there an interplay between Total Antioxidant Capacity and increase of oxidative stress levels? The authors should add other information about this phenomenon, especially improve the clinical meaning and utility of TAC.
Answer: We previously deeply discussed about the clinical meaning of the TAC value and our thoughts are reported in the ref. 13 (Antioxidants 2021; DOI: 10.3390/antiox10010058). In this work, the shift from the TAC to the chemical speciation model is properly useful to overcome the limits found out in the TAC by scientists over time.
The discussion about the clinical meaning of the TAC was no more proposed in this work to avoid repetitions, but the ref. 13 was moved to the beginning of the Introduction, to make easier for the readers follow the topic (lines 40-41).
Why the choice of this category of patients? Could it depend by disease?
Answer: The group of pathological subjects was chosen selecting a condition probably involving an unbalance of the oxidative status of plasma with the aim to test the speciation model under development. The reason of the choice was inserted in the Introduction (lines 78-80).
Plasma is the target of this investigation. It is mostly water, and contains important proteins, glucose, clotting factors, electrolytes, hormones, vitamins, oxidants, carbon dioxide and oxygen. In this context, it plays a vital role for measuring the TAC efficiency. However, some of these molecules (vitamins or oxidants), transported by the blood stream, could interact with erythrocytes plasma membrane structures, thus causing potential consequences on erythrocytes (the most numerous cells in the blood) . How do the authors explain this? Could the measure be changed? The organism aims to maintain a beneficial chemical equilibrium to perform specific biological functions, being a method depending quantity, the concentration of the plasma redox active biomolecules could be altered? I suggest to add this aspect in your study, and to examine the following papers (10.3390/antiox9010025 et al.,), in which the authors underscore the role of different substance in erythrocytes homeostasis, as well as the potential antioxidant effects. It is possible that there is not a direct ratio between TAC efficiency and specific disease. In fact, the TAC is major in cardiopathic individuals compared to the control, and these findings could explain the capacity of the organism to defend by reactive species. The authors should also analyze this aspect.
Answer: Our study deals with the extracellular antioxidant capacity, as clarified in the introduction section. The involvement of the cellular components of blood was not taken into account. See also a previous paper published on Antioxidants (DOI: 10.3390/antiox10010058) and now mentioned in the text as ref. 3 (ref. 13, original version). The reference suggested by the reviewer is very interesting, but it does not apply to this study.
Plasma from 25 cardiovascular surgery patients –17 males and 8 females– was collected and subjected to the measurements, data treatments and modelling as those of the healthy individuals (85 blood donors – 47 males and 38 females). Is this the correct number? The number of cardiovascular surgery patients could be very low compared to healthy volunteers in order to analysis. The authors should explain the choice.
Answer: We understand the reviewer’s remark, since the number of the cardiovascular surgery patients is quite low. We were first focused on establishing the reference of the study using a wide number of blood donors to build the chemical model on the dataset of healthy individuals. Afterwards, we started to test the reliability of our speciation model on a small group of pathological subjects bearing a disease probably involving an unbalance of the oxidative status of plasma. We thus verified that our chemical model responds to biological variability, even if on a small number of cases.
The paper's approach is methodological. The approach set up here can be applied later to other pathologies, and strengthened in its analytical and forecasting capabilities. Such methodology could be useful in clinical chemistry to attribute a clinical meaning to TAC values.
A text was added in the Conclusions paragraph to strengthen the concept (lines 486 -489 and 503-504).
Reviewer 3 Report
The manuscript by Presnetti et al. entitled Biomolecules responsible of [sic] the TAC… defines the source(s), i.e., metabolic, dietary, of total antioxidant capacity and redox active molecules in healthy and diseased individuals to develop a chemical speciation model.
There are a few points of clarification to consider:
In the title, should it be responsible “for” the TAC instead of “of?”
The manuscript requires another round of critical proofreading. Several paragraphs/sentences can be merged (lines 106-112 or lines 115-126).
Could the authors clarify the term hyperactive free radical (line 38) compared to reactive as used in ROS?
On line 175, could DTNB be defined as (5,5-dithio-bis-(2-nitrobenzoic acid; Ellman’s reagent)?
Was this study approved by an Institutional Review Board (IRB)? If so, could a statement reflecting this be added?
Would it flow better in the tables, to relocate the standard deviation column next to the mean column? This is at the discretion of the authors.
Could the phrase on line 346 (“in the Supplementary Materials file” be move to first mention on line 331 for clarity?
On line 440, consideration should be given to replacing the term newsworthy. Perhaps, “to draw some biologically meaningful conclusions?”
Author Response
Comments and Suggestions for Authors
The manuscript by Presnetti et al. entitled Biomolecules responsible of [sic] the TAC… defines the source(s), i.e., metabolic, dietary, of total antioxidant capacity and redox active molecules in healthy and diseased individuals to develop a chemical speciation model.
There are a few points of clarification to consider:
In the title, should it be responsible “for” the TAC instead of “of?”
Answer: The title was changed.
The manuscript requires another round of critical proofreading. Several paragraphs/sentences can be merged (lines 106-112 or lines 115-126).
Answer: The indicated lines deals with chemicals and apparatuses and are already expressed in a short form; therefore, we did not understand what the reviewer want to communicate us because we do not find repetitions.
Could the authors clarify the term hyperactive free radical (line 38) compared to reactive as used in ROS?
Answer: Line 38: the term “hyperactive” was removed.
On line 175, could DTNB be defined as (5,5-dithio-bis-(2-nitrobenzoic acid; Ellman’s reagent)?
Answer: Now lines 176 - 177: DTNB was specified as requested.
Was this study approved by an Institutional Review Board (IRB)? If so, could a statement reflecting this be added?
Answer: In the paragraph “2.4 Subjects” the details about the experimental design are reported. We have already discussed ethical issues with the Special Issue Editor and we agreed the study does not need the ethical committee evaluation. This is because plasma samples were always collected during routine sessions, scheduled for purposes unrelated to the study. The study simply used leftovers of plasma samples accompanied with anonymized data (investigators only knew gender and age). No one was asked for any kind of preparation, nor was the administration of anything specific, nor were there any special requests related to our study. A simple data collection was made on waste samples to characterize the redox chemistry of plasma without diagnostic or therapeutic goals.
Would it flow better in the tables, to relocate the standard deviation column next to the mean column? This is at the discretion of the authors.
Answer: Tables 1 and 2: the columns of “mean” and “median” were inverted to meet the reviewer’s indication.
Could the phrase on line 346 (“in the Supplementary Materials file” be move to first mention on line 331 for clarity?
Answer: Now line 353: the text “in the Supplementary Materials file” was removed.
On line 440, consideration should be given to replacing the term newsworthy. Perhaps, “to draw some biologically meaningful conclusions?”
Answer: Now line 465: the term “newsworthy” was removed.
Round 2
Reviewer 2 Report
Accept in present form
